Mutations in OAS1 genes

ABSTRACT

Modified amino acid sequences of OAS1 proteins in non-human primates, and genes related thereto, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S patent application Ser.No. 13/180,132 filed Jul. 11, 2011, which is a continuation of U.S.patent application Ser. No. 12/248,810 filed Oct. 9, 2008, now U.S. Pat.No. 8,030,046 which claims the benefit of priority from U.S. patentapplication Ser. No. 11/416,790 filed May 3, 2006, now abandoned whichclaims benefit from U.S. Provision Patent Application titled MUTATION INOAS1 GENES Ser. No. 60/677,680 filed May 4,2005 under 35 U.S.C §119. Theforegoing patent applications are incorporated herein by reference intheir entirety.

TECHNICAL FIELD

The present invention relates to a method for detecting a mutation in ahuman or non-human primate oligoadenylate synthetase gene, and to OAS1proteins having at least one amino acid modification.

BACKGROUND OF THE INVENTION

A number of diseases have been identified to date in which naturalresistance to infection exists in the human population. Alter and Moyer,J. Acquir. Immune Defic. Syndr. Hum Retrovirol. 18 Suppl. 1:S6-10 (1998)report hepatitis C viral infection (HCV) rates as high as 90% inhigh-risk groups such as injecting drug users. However, the mechanism bywhich the remaining 10% are apparently resistant to infection has notbeen identified in the literature. Proteins that play a role in HCVinfection include the 2-prime, 5-prime oligoadenylate synthetases. OASsare interferon-induced proteins characterized by their capacity tocatalyze the synthesis of 2-prime, 5-prime oligomers of adenosine(2-5As). Hovanessian et al., EMBO 6: 1273-1280 (1987) found thatinterferon-treated human cells contain several OASs corresponding toproteins of 40 (OAS1), 46 (OAS1), 69, and 100 kD. Marie et al., Biochem.Biophys. Res. Commun. 160:580-587 (1989) generated highly specificpolyclonal antibodies against p69, the 69-kD OAS. By screening aninterferon-treated human cell expression library with the anti-p69antibodies, Marie and Hovanessian, J. Biol. Chem. 267: 9933-9939 (1992)isolated a partial OAS2 cDNA. They screened additional libraries withthe partial cDNA and recovered cDNAs encoding two OAS2 isoforms. Thesmaller isoform is encoded by two mRNAs that differ in the length of the3-prime untranslated region.

Northern blot analysis revealed that OAS2 is expressed as fourinterferon-induced mRNAs in human cells. The predicted OAS2 proteinshave a common 683-amino acid sequence and different 3-prime termini.According to SDS-PAGE of in vitro transcription/translation products,two isoforms have molecular masses of 69 and 71 kD. Both isoformsexhibited OAS activity in vitro. Sequence analysis indicated that OAS2contains two OAS1-homologous domains separated by a proline-richputative linker region. The N- and C-terminal domains are 41% and 53%identical to OAS1, respectively.

By fluorescence in situ hybridization and by inclusion within mappedclones, Hovanian et al., Genomics 52: 267-277 (1998) determined that theOAS1, OAS2, and OAS3 genes are clustered with a 130-kb region on12q24.2. 2-5As bind to and activate RNase I, which degrades viral andcellular RNAs, leading to inhibition of cellular protein synthesis andimpairment of viral replication.

A fourth human OAS gene, referred to as OASL, differs from OAS1, OAS2and OAS3 in that OASL lacks enzyme activity. The OASL gene encodes atwo-domain protein composed of an OAS unit fused to a 164 amino acidC-terminal domain that is homologous to a tandem repeat of ubiquitin.(Eskildsen et al., Nuc. Acids Res. 31:3166-3173, 2003; Kakuta et al., J.Interferon & Cytokine Res. 22:981-993, 2002.)

Because of their role in inhibiting viral replication and viralinfection, there is a need in the art for methods and compositions thatsuppress viral replication related to OAS1 activity, including aprofound need for inhibitor-based therapies that suppress HCVreplication.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to detecting hepatitis Cresistance-related mutations which may be characterized as mutations inthe oligoadenylate synthetase 1 gene.

In one embodiment, a genetic screening method is contemplated. Themethod comprises assaying a nucleic acid sample isolated from a human ornon-human primate for the presence of an oligoadenylate synthetase 1gene mutation causing an amino acid modification at one or more ofpositions 1, 24, 25, 28, 31, 36, 47, 53, 54, 64, 69, 74, 104, 108, 112,113, 114, 115, 116, 117, 118, 119, 127, 130, 139, 142, 160, 161, 162,166, 175, 179, 226, 242, 246, 248, 250, 254, 274, 279, 282, 284, 288,289, 292, 295, 314, 315, and 335 for all oligoadenylate synthetase 1(OAS1) forms (including without limitation SEQ ID NO:1).

In a further embodiment, a genetic screening method is contemplated. Themethod comprises assaying a nucleic acid sample isolated from a human ornon-human primate for the presence of an oligoadenylate synthetase 1gene mutation causing an amino acid modification at position 363 for alloligoadenylate synthetase 1 forms that are carboxyl-terminus homologousto Genbank accession NP_(—)002525.1 (including without limitation SEQ IDNO:3).

In a yet further embodiment, a genetic screening method is contemplated.The method comprises assaying a nucleic acid sample isolated from ahuman or non-human primate for the presence of an oligoadenylatesynthetase 1 gene mutation causing an amino acid modification at one ormore of amino acid positions 347, 350, 352, 353, 354, 356, 357, 361,363, 364, 365, 369, 371, 373, 374, 375, 378, 379, 382, 388, 389, or 394for all oligoadenylate synthetase 1 forms that are carboxyl-terminushomologous to Genbank accession NP_(—)058132.1 (including withoutlimitation SEQ ID NO:2).

In a yet further embodiment, a genetic screening method is contemplated.The method comprises assaying a nucleic acid sample isolated from ahuman or non-human primate for the presence of an oligoadenylatesynthetase 1 gene mutation causing an amino acid modification at one ormore of amino acid positions 347, 361, 364, 372, 384, 385, or 399 forall oligoadenylate synthetase 1 forms that are carboxyl-terminushomologous to Genbank accession NP_(—)001027581.1 (including withoutlimitation SEQ ID NO:4).

In a further embodiment, the invention provides a protein having atleast one amino acid modification at positions 1, 24, 25, 28, 31, 36,47, 53, 54, 64, 69, 74, 104, 108, 112, 113, 114, 115, 116, 117, 118,119, 127, 130, 139, 142, 160, 161, 162, 166, 175, 179, 226, 242, 246,248, 250, 254, 274, 279, 282, 284, 288, 289, 292, 295, 314, 315, and 335for all oligoadenylate synthetase 1 (OAS1) forms (including withoutlimitation SEQ ID NO:1), and use of the protein to prepare a diagnosticfor resistance to viral infection, preferably flaviviral infection, mostpreferably hepatitis C infection. In specific embodiments, thediagnostic is an antibody.

In a further embodiment, the invention provides a OAS1 protein having anamino acid modification at position 363 for all oligoadenylatesynthetase 1 forms that are carboxyl-terminus homologous to Genbankaccession NP_(—)002525.1 (including without limitation SEQ ID NO:3), anduse of the protein to prepare a diagnostic for resistance to viralinfection, preferably flaviviral infection, most preferably hepatitis Cinfection. In specific embodiments, the diagnostic is an antibody.

In a further embodiment, the invention provides a OAS1 protein having atleast one amino acid modification at positions 347, 350, 352, 353, 354,356, 357, 361, 363, 364, 365, 369, 371, 373, 374, 375, 378, 379, 382,388, 389, and 394 for all oligoadenylate synthetase 1 forms that arecarboxyl-terminus homologous to Genbank accession NP_(—)058132.1(including without limitation SEQ ID NO:2) and use of the protein toprepare a diagnostic for resistance to viral infection, preferablyflaviviral infection, most preferably hepatitis C infection. In specificembodiments, the diagnostic is an antibody.

In a yet further embodiment, the invention provides a OAS1 proteinhaving at least one amino acid modification at positions 347, 361, 364,372, 384, 385, or 399 for all oligoadenylate synthetase 1 forms that arecarboxyl-terminus homologous to Genbank accession NP_(—)001027581.1(including without limitation SEQ ID NO: 4) and use of the protein toprepare a diagnostic for resistance to viral infection, preferablyflaviviral infection, most preferably hepatitis C infection. In specificembodiments, the diagnostic is an antibody.

In a still further embodiment, the invention provides a therapeuticcompound for preventing or inhibiting infection by a virus, preferably aflavivirus, most preferably hepatitis C virus, wherein the therapeuticcompound is a protein having at least one amino acid modificationaccording to the invention. In other embodiments the therapeuticcompound is a polynucleotide, such as DNA or RNA, encoding the protein.

In a still further embodiment, the invention provides a therapeuticcompound for preventing or inhibiting infection by a virus, preferably aflavivirus, most preferably a hepatitis C virus, wherein the therapeuticcompound is a protein encoded by OAS1 of the invention having one ormore of the disclosed amino acid modifications.

In a still further embodiment, the invention provides a therapeuticcompound for preventing or inhibiting infection by a virus, preferably aflavivirus, most preferably hepatitis C virus, wherein the therapeuticcompound mimics the beneficial effects of at least one mutation of theinvention. The therapeutic compound can be a small molecule, protein,peptide, DNA or RNA molecule, or antibody.

In a still further embodiment, the invention provides a therapeuticcompound for preventing or treating cancer, preferably prostate cancer,wherein the therapeutic compound is a protein encoded by an OAS1 genehaving at least one mutation of the invention. In other embodiments thetherapeutic compound is a polynucleotide, such as DNA or RNA, encodingthe protein.

In a still further embodiment, the invention provides a therapeuticcompound for preventing or treating cancer, preferably prostate cancer,wherein the therapeutic compound is a OAS1 protein having at least oneamino acid modification of the invention:

In a still further embodiment, the invention provides a therapeuticcompound for preventing or treating cancer, preferably prostate cancer,wherein the therapeutic compound mimics the beneficial effects of atleast one mutation of the invention. The therapeutic compound can be asmall molecule, protein, peptide, DNA or RNA molecule, or antibody.

In further embodiments, the therapeutic compound is capable ofinhibiting the activity of OAS1 or at least one sub-region orsub-function of the entire protein, and such compounds are representedby antisense molecules, ribozymes, and RNAi molecules capable ofspecifically binding to OAS1 polynucleotides, and by antibodies andfragments thereof capable of specifically binding to OAS1 proteins andpolypeptides.

The present invention provides, in another embodiment, inhibitors ofOAS1. Inventive inhibitors include, but are not limited to, antisensemolecules, ribozymes, RNAi, antibodies or antibody fragments, proteinsor polypeptides as well as small molecules. Exemplary antisensemolecules comprise at least 10, 15 or 20 consecutive nucleotides of, orthat hybridize under stringent conditions to the polynucleotide encodingOAS1 having at least one amino acid modification of the invention.

In a still further embodiment, inhibitors of OAS1 are envisioned thatspecifically bind to the region of the protein defined by a OAS1polypeptide having an amino acid modification of the invention.Inventive inhibitors include but are not limited to antibodies, antibodyfragments, small molecules, proteins, or polypeptides.

In a still further embodiment, inhibitors of OAS1 are envisioned thatare comprised of antisense or RNAi molecules that specifically bind orhybridize to a polynucleotide encoding an OAS1 protein having at leastone amino acid modification of the invention.

In further embodiments, compositions are provided that comprise one ormore OAS1 inhibitors in a pharmaceutically acceptable carrier.

Additional embodiments provide methods of decreasing OAS1 geneexpression or biological activity.

Additional embodiments provide for methods of specifically increasing ordecreasing the expression of certain forms of the OAS1 gene having atleast one mutation as disclosed by the invention.

The invention provides an antisense oligonucleotide comprising at leastone modified internucleoside linkage.

The invention further provides an antisense oligonucleotide having aphosphorothioate linkage.

The invention still further provides an antisense oligonucleotidecomprising at least one modified sugar moiety.

The invention also provides an antisense oligonucleotide comprising atleast one modified sugar moiety which is a 2′-O-methyl sugar moiety.

The invention further provides an antisense oligonucleotide comprisingat least one modified nucleobase.

The invention still further provides an antisense oligonucleotide havinga modified nucleobase wherein the modified nucleobase is5-methylcytosine.

The invention also provides an antisense compound wherein the antisensecompound is a chimeric oligonucleotide.

The invention provides a method of inhibiting the expression of humanOAS1 in human cells or tissues comprising contacting the cells ortissues in vivo with an antisense compound or a ribozyme of 8 to 35nucleotides in length targeted to a nucleic acid molecule encoding humanOAS1 so that expression of human OAS1 is inhibited.

The invention further provides a method of decreasing or increasingexpression of specific forms of OAS1 in vivo, such forms being definedby having at least one mutation at a position according to theinvention, using antisense or RNAi compounds or ribozymes.

The invention further provides a method of modulating growth of cancercells comprising contacting the cancer cells in vivo with an antisensecompound or ribozyme of 8 to 35 nucleotides in length targeted to anucleic acid molecule encoding human OAS1 so that expression of humanOAS1 is inhibited.

The invention still further provides for identifying target regions ofOAS1 polynucleotides. The invention also provides labeled probes foridentifying OAS1 polynucleotides by in situ hybridization.

The invention provides for the use of an OAS1 inhibitor according to theinvention to prepare a medicament for preventing or inhibiting HCVinfection.

The invention further provides for directing an OAS1 inhibitor tospecific regions of the OAS1 protein or at specific functions of theprotein.

The invention also provides a pharmaceutical composition for inhibitingexpression of OAS1, comprising an antisense oligonucleotide according tothe invention in a mixture with a physiologically acceptable carrier ordiluent.

The invention further provides a ribozyme capable of specificallycleaving OAS1 RNA, and a pharmaceutical composition comprising theribozyme.

The invention also provides small molecule inhibitors of OAS1 whereinthe inhibitors are capable of reducing the activity of OAS1 or ofreducing or preventing the expression of OAS1 mRNA.

The invention further provides for inhibitors of OAS1 that modifyspecific functions of the protein other than the synthesis of 2′-5′oligoadenylates, such functions including interaction with otherproteins such as Hepatitis C virus NS5A protein.

The invention further provides for compounds that alterpost-translational modifications of OAS1 including but not limited toglycosylation and phosphorylation.

The invention further provides a human genetic screening method foridentifying an oligoadenylate synthetase gene mutation comprising: (a)treating, under amplification conditions, a sample of genomic DNA from ahuman with a polymerase chain reaction (PCR) primer pair for amplifyinga region of human genomic DNA containing at least one mutation of anOAS1 gene according to the invention, said treating producing anamplification product containing said region; and (b) detecting in theamplification product of step (a) the presence of a nucleotide mutationat a nucleotide position of the invention, thereby identifying saidmutation.

The invention also relates to a method for detecting in a human ahepatitis C infection resistance disease allele containing a mutationcomprising substitution of a non wild-type nucleotide for a wild-typenucleotide at a nucleotide position corresponding to an amino acidmodification of the invention in the OAS1 protein encoded by the gene ofoligoadenylate synthetase gene (OAS1), which method comprises: (a)forming a polymerase chain reaction (PCR) admixture by combining, in aPCR buffer, a sample of genomic DNA from said human and anoligoadenylate synthetase gene-specific PCR primer pair; (b) subjectingthe PCR admixture to a plurality of PCR thermocycles to produce anoligoadenylate synthetase gene amplification product; and (c) treating,under hybridization conditions products produced in step (b), with aprobe capable of detecting said mutation.

Also provided is an isolated OAS1 inhibitor selected from the groupconsisting of an antisense oligonucleotide, a ribozyme, a smallinhibitory RNA (RNAi), a protein, a polypeptide, an antibody, and asmall molecule. The isolated inhibitor may be an antisense molecule orthe complement thereof comprising at least 15 consecutive nucleic acidsof a polynucleotide sequence corresponding to a OAS1 gene mutationassociated with an amino acid substitution of the invention.

The isolated OAS1 inhibitor may be selected from the group consisting ofan antibody and an antibody fragment. Also provided is a compositioncomprising a therapeutically effective amount of at least one OAS1inhibitor in a pharmaceutically acceptable carrier.

The invention also relates to a method of inhibiting the expression ofOAS1 in a mammalian cell, comprising administering to the cell an OAS1inhibitor selected from the group consisting of an antisenseoligonucleotide, a ribozyme, a protein, an RNAi, a polypeptide, anantibody, and a small molecule.

The invention further relates to a method of inhibiting the expressionof OAS1 gene expression in a subject, comprising administering to thesubject, in a pharmaceutically effective vehicle, an amount of anantisense oligonucleotide which is effective to specifically hybridizeto all or part of a selected target nucleic acid sequence derived fromsaid OAS1 gene.

The invention still further relates to a method of preventing infectionby a flavivirus in a human subject susceptible to the infection,comprising administering to the human subject an OAS1 inhibitor selectedfrom group consisting of an antisense oligonucleotide, a ribozyme, anRNAi, a protein, a polypeptide, an antibody, and a small molecule,wherein said OAS1 inhibitor prevents infection by said flavivirus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an amino acid sequence of a therapeutic form of OAS1 protein(SEQ ID NO:1).

FIG. 2 is a table listing amino acid substitutions useful in alltherapeutic forms of OAS1.

FIG. 3 is a table listing primate OAS1 amino acid modifications usefulin therapeutic forms of OAS1. Positions indicated with * refer to formsof OAS1 that are carboxyl-terminus homologous to Genbank accessionNP_(—)002525.1. Positions indicated with +refer to forms of OAS1 thatare carboxyl-terminus homologous to Genbank accession NP_(—)0581321.Positions indicated with ^ refer to forms of OAS1 that arecarboxyl-terminus homologous to Genbank accession NP_(—)001027581.1.

FIG. 4 is a chart indicating the positions of mutations of primate OAS1genes and corresponding amino acid modifications. Position 3916008corresponds to amino acid variants SerllePheArgGluVal (SEQ ID NO:18) andArgAlaPheSerValLysPhe (SEQ ID NO:19).

FIG. 5 is a listing of additional OAS1 isoforms of the presentinvention, including human and non-human primate forms. Also providedare mutations of the primate isoforms. These isoforms, either alone ortogether with any mutations identified in the present invention, areuseful for the diagnostic, therapeutic, and other purposes describedherein.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to novel mutations in the oligoadenylatesynthetase 1 gene, use of these mutations for diagnosis ofsusceptibility or resistance to viral infection, to proteins encoded bya gene having a mutation according to the invention, and to preventionor inhibition of viral infection using the proteins, antibodies, andrelated nucleic acids. These mutations correlate with resistance of thecarrier to infection with flavivirus, particularly hepatitis C virus.

Much of current medical research is focused on identifying mutations anddefects that cause or contribute to disease. Such research is designedto lead to compounds and methods of treatment aimed at the diseasestate. Less attention has been paid to studying the genetic influencesthat allow people to remain healthy despite exposure to infectiousagents and other risk factors. The present invention represents asuccessful application of a process developed by the inventors by whichspecific populations of human subjects are ascertained and analyzed inorder to discover genetic variations or mutations that confer resistanceto disease. The identification of a sub-population segment that has anatural resistance to a particular disease or biological conditionfurther enables the identification of genes and proteins that aresuitable targets for pharmaceutical intervention, diagnostic evaluation,or prevention, such as prophylactic vaccination.

A sub-population segment was previously identified and disclosed inco-pending application Ser. No. 10/972,135 and was comprised ofindividuals who, despite repeated exposure to hepatitis C virus (HCV)have nonetheless remained sero-negative, while cohorts have becomeinfected (sero-positive). The populations studied included hemophiliacpatients subjected to repeated blood transfusions, and intravenous drugusers who become exposed through shared needles and other risk factors.

The present disclosure provides mutations identified in OAS1 genes ofnon-human primates, as described in Example 1.

Application Ser. No.10/972,135 provides disclosure related to HCVinfection; definitions; modes of practicing the invention;polynucleotide analysis; preparation of polynucleotide primers;polymerase chain reaction; nucleic acid sequence analysis; detection ofmembrane-immobilized target sequences; scanning techniques for detectionof base substitutions; therapeutic agents for restoring and/or enhancingOAS1 function; therapeutic agents for inhibition of OAS1 function;ribozymes; RNAi; proteins and polypeptides; small molecules; methods forassessing the efficacy of OAS1 inhibitors; and pharmaceuticalcompositions. Application Ser. No.10/972,135 is hereby incorporatedherein by reference in its entirety.

The polypeptides of the present invention are able, as part of theirnative function, to transduce across a cell membrane and mediate theirantiviral effects in the absence of a delivery vector or expressionvehicle. The cell transduction properties of basic, positively chargedproteins has been previously described and is well known to thoseskilled in the art (Ryser and Hancock, Science. 1965 Oct22;150(695):501-3).

In the case where the polypeptides are prepared as a liquid formulationand administered by injection, preferably the solution is an isotonicsalt solution containing 140 millimolar sodium chloride and 10millimolar calcium at pH 7.4. The injection may be administered, forexample, in a therapeutically effective amount, preferably in a dose ofabout 1 μg/kg body weight to about 5 mg/kg body weight daily, takinginto account the routes of administration, health of the patient, etc.

The polypeptide(s) of the present invention may be employed incombination with a suitable pharmaceutical carrier. Such compositionscomprise a therapeutically effective amount of the protein, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

The polypeptide(s) of the present invention can also be modified bychemically linking the polypeptide to one or more moieties or conjugatesto enhance the activity, cellular distribution, or cellular uptake ofthe polypeptide(s). Such moieties or conjugates include lipids such ascholesterol, cholic acid, thioether, aliphatic chains, phospholipids andtheir derivatives, polyamines, polyethylene glycol (PEG), palmitylmoieties, and others as disclosed in, for example, U.S. Pat. Nos.5,514,758, 5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371,5,597,696 and 5,958,773.

The polypeptides of the present invention may also be modified to targetspecific cell types for a particular disease indication, including butnot limited to liver cells in the case of hepatitis C infection. As canbe appreciated by those skilled in the art, suitable methods have beendescribed that achieve the described targeting goals and include,without limitation, liposomal targeting, receptor-mediated endocytosis,and antibody-antigen binding. In one embodiment, the asiaglycoproteinreceptor may be used to target liver cells by the addition of agalactose moiety to the polypeptide(s). In another embodiment, mannosemoieties may be conjugated to the polypeptide(s) in order to target themannose receptor found on macrophages and liver cells. As one skilled inthe art will recognize, multiple delivery and targeting methods may becombined. For example, the polypeptide(s) of the present invention maybe targeted to liver cells by encapsulation within liposomes, suchliposomes being conjugated to galactose for targeting to theasialoglycoprotein receptor.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptide of the present invention may be employed in conjunction withother therapeutic compounds.

When the polypeptide(s) of the present invention are used as apharmaceutical, they can be given to mammals, in a suitable vehicle.When the polypeptides of the present invention are used as apharmaceutical as described above, they are given, for example, intherapeutically effective doses of about 10 μg/kg body weight to about10 mg/kg body weight daily, taking into account the routes ofadministration, health of the patient, etc. The amount given ispreferably adequate to achieve prevention or inhibition of infection bya virus, preferably a flavivirus, most preferably RSV and HCV,prevention or treatment of cancer, inflammation, diabetes, or otherdiseases.

The proteins, their fragments or other derivatives, or analogs thereof,or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal,monoclonal, chimeric, single chain, Fab fragments, or the product of anFab expression library. Various procedures known in the art may be usedfor the production of polyclonal antibodies.

Antibodies generated against the polypeptide(s) of the present inventioncan be obtained by direct injection of the polypeptide into an animal orby administering the polypeptide to an animal, preferably a nonhuman.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies binding the whole native polypeptide.Moreover, a panel of such antibodies specific to a large number ofpolypeptides can be used.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-597), the trioma technique, the human B-cell hybridomatechnique (Kozbor, et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Coe, etal., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention.

The antibodies can be used in methods relating to the localization andactivity of the protein sequences of the invention, e.g., for imagingthese proteins, measuring levels thereof in appropriate physiologicalsamples, and the like.

The invention provides for polypeptides that differ from thepolypeptides of FIGS. 1-5 by 1 to 34 amino acids, such differences mayinclude substitutions, insertions, deletions, the incorporation ofmodified amino acids or amino acid derivatives, and the addition ordeletion of amino acids from the C-terminus or N-terminus of thepolypeptides. The invention provides for therapeutic and prophylacticuses of these polypeptides including but not limited to the treatment ofvirus infection, neoplasm, cancer, diabetes, and to promote cell growthand differentiation and tissue regeneration. The invention provides forpolynucleotides encoding the polypeptides of the invention and usesthereof including but not limited to uses in manufacturing thepolypeptides, as gene therapies, as diagnostic tools, etc.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions of the polypeptidesas active ingredients for a therapeutic application. These compositionscan also be used in the method of the present invention. In general thepharmaceutical composition for inhibiting virus infection, cancer,neoplasm, inflammation, or other disease in a mammal or subject includesan effective amount of at least one polypeptide as described aboveneeded for the practice of the invention, or a fragment thereof shown tohave the same effect, and a pharmaceutically physiologically acceptablecarrier or diluent. According to the present invention, a pharmaceuticalcomposition can be composed of two or more of the polypeptides of FIGS.1-5 in combination. The pharmaceutical composition may further becomposed of a single polypeptide that contains one or more of themodifications of FIGS. 1-5 within a contiguous molecule.

The compositions can be administered orally, subcutaneously, orparenterally including intravenous, intraarterial, intramuscular,intraperitoneally, and intranasal administration, as well as intrathecaland infusion techniques as required. The pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles as well as implant carriersgenerally refer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention. Cationic lipids may also be included in the composition tofacilitate polypeptide uptake. Implants of the compounds are alsouseful. In general, the pharmaceutical compositions are sterile.

The present invention relates to compositions of the polypeptides towhich a detectable label is attached, such as a fluorescent,chemiluminescent or radioactive molecule.

Another example is a pharmaceutical composition which may be formulatedby known techniques using known materials, see, Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa. 18042) pp. 1435-1712, which are herein incorporated by reference.Generally, the formulation will depend on a variety of factors such asadministration, stability, production concerns and other factors. Thepolypeptides of FIGS. 1-5 may be administered by injection or bypulmonary administration via inhalation. Enteric dosage forms may alsobe available, and therefore oral administration may be effective. Thepolypeptides of the invention may be inserted into liposomes or othermicrocarriers for delivery, and may be formulated in gels or othercompositions for sustained release. Although preferred compositions willvary depending on the use to which the composition will be put,generally, for the polypeptides of the present invention, preferredpharmaceutical compositions are those prepared for subcutaneousinjection or for pulmonary administration via inhalation, although theparticular formulations for each type of administration will depend onthe characteristics of the specific polypeptide.

Therapeutic formulations of the polypeptides or polypeptide conjugatesof the invention are typically administered in a composition thatincludes one or more pharmaceutically acceptable carriers or excipients.Such pharmaceutical compositions may be prepared in a manner known perse in the art to result in a polypeptide pharmaceutical that issufficiently storage-stable and is suitable for administration to humansor animals.

The polypeptides or polypeptide conjugates of the invention can be used“as is” and/or in a salt form thereof. Suitable salts include, but arenot limited to, salts with alkali metals or alkaline earth metals, suchas sodium, potassium, calcium and magnesium, as well as e.g. zinc salts.These salts or complexes may by present as a crystalline and/oramorphous structure.

“Pharmaceutically acceptable” means a carrier or excipient that at thedosages and concentrations employed does not cause any untoward effectsin the patients to whom it is administered. Such pharmaceuticallyacceptable carriers and excipients are well known in the art (seeRemington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed.,Mack Publishing Company (1990); Pharmaceutical Formulation Developmentof Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor &Francis (2000); and Handbook of Pharmaceutical Excipients, 3rd edition,A. Kibbe, Ed., Pharmaceutical Press (2000)).

The composition of the invention may be administered alone or inconjunction with other therapeutic agents. Ribavirin and interferonalpha, for example, have been shown to be an effective treatment for HCVinfection when used in combination. Their efficacy in combinationexceeds the efficacy of either drug product when used alone. Thecompositions of the invention may be administered alone or incombination with interferon, ribavirin and/or a variety of smallmolecules that are being developed against both viral targets (viralproteases, viral polymerase, assembly of viral replication complexes)and host targets (host proteases required for viral processing, hostkinases required for phosphorylation of viral targets such as NS5A andinhibitors of host factors required to efficiently utilize the viralIRES). Cytokines may be co-administered, such as for example IL-2,IL-12, IL-23, IL-27, or IFN-gamma. These agents may be incorporated aspart of the same pharmaceutical composition or may be administeredseparately from the polypeptides or conjugates of the invention, eitherconcurrently or in accordance with another treatment schedule. Inaddition, the polypeptides, polypeptide conjugates or compositions ofthe invention may be used as an adjuvant to other therapies.

A “patient” for the purposes of the present invention includes bothhumans and other mammals. Thus the methods are applicable to both humantherapy and veterinary applications

The pharmaceutical composition comprising the polypeptide or conjugateof the invention may be formulated in a variety of forms, e.g. as aliquid, gel, lyophilized, or as a compressed solid. The preferred formwill depend upon the particular indication being treated and will beapparent to one skilled in the art.

The administration of the formulations of the present invention can beperformed in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,intrathecally, vaginally, rectally, intraocularly, or in any otheracceptable manner. The formulations can be administered continuously byinfusion, although bolus injection is acceptable, using techniques wellknown in the art, such as pumps (e.g., subcutaneous osmotic pumps) orimplantation. In some instances the formulations may be directly appliedas a solution or spray.

An example of a pharmaceutical composition is a solution designed forparenteral administration. Although in many cases pharmaceuticalsolution formulations are provided in liquid form, appropriate forimmediate use, such parenteral formulations may also be provided infrozen or in lyophilized form. In the former case, the composition mustbe thawed prior to use. The latter form is often used to enhance thestability of the active compound contained in the composition under awider variety of storage conditions, as it is recognized by thoseskilled in the art that lyophilized preparations are generally morestable than their liquid counterparts. Such lyophilized preparations arereconstituted prior to use by the addition of one or more suitablepharmaceutically acceptable diluents such as sterile water for injectionor sterile physiological saline solution.

Parenterals may be prepared for storage as lyophilized formulations oraqueous solutions by mixing, as appropriate, the polypeptide having thedesired degree of purity with one or more pharmaceutically acceptablecarriers, excipients or stabilizers typically employed in the art (allof which are termed “excipients”), for example buffering agents,stabilizing agents, preservatives, isotonifiers, non-ionic detergents,antioxidants and/or other miscellaneous additives.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are typically present at a concentrationranging from about 2 mM to about 50 mM. Suitable buffering agents foruse with the present invention include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-trisodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additional possibilities are phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and are typicallyadded in amounts of about 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides (e.g. benzalkonium chloride, bromide oriodide), hexamethonium chloride, alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are added to ensure isotonicity of liquid compositionsand include polyhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol. Polyhydric alcohols can be present in an amountbetween 0.1% and 25% by weight, typically 1% to 5%, taking into accountthe relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, omithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol,alpha-monothioglycerol and sodium thiosulfate; low molecular weightpolypeptides (i.e. <10 residues); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; monosaccharides such as xylose, mannose,fructose and glucose; disaccharides such as lactose, maltose andsucrose; trisaccharides such as raffinose, and polysaccharides such asdextran. Stabilizers are typically present in the range of from 0.1 to10,000 parts by weight based on the active protein weight.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe present to help solubilize the therapeutic agent as well as toprotect the therapeutic polypeptide against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stress without causing denaturation of the polypeptide. Suitablenon-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers(184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.).

Additional miscellaneous excipients include bulking agents or fillers(e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g.,ascorbic acid, methionine, vitamin E) and cosolvents.

The active ingredient may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences, supra.

In one aspect of the invention the composition is a liquid composition,such as an aqueous composition, and comprises a sulfoalkyl ethercyclodextrin derivative.

Parenteral formulations to be used for in vivo administration must besterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing thepolypeptide or conjugate, the matrices having a suitable form such as afilm or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the ProLease® technology orLupron Depot@ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for long periods such asup to or over 100 days, certain hydrogels release proteins for shortertime periods. When encapsulated polypeptides remain in the body for along time, they may denature or aggregate as a result of exposure tomoisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization depending on the mechanism involved. For example, ifthe aggregation mechanism is discovered to be intermolecular S-S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulthydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Oral administration of the peptides and peptide conjugates is anintended practice of the invention. For oral administration, thepharmaceutical composition may be in solid or liquid form, e.g. in theform of a capsule, tablet, suspension, emulsion or solution. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a given amount of the active ingredient. A suitabledaily dose for a human or other mammal may vary widely depending on thecondition of the patient and other factors, but can be determined bypersons skilled in the art using routine methods.

Solid dosage forms for oral administration may include capsules,tablets, suppositories, powders and granules. In such solid dosageforms, the active compound may be admixed with at least one inertdiluent such as sucrose, lactose, or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances, e.g. lubricatingagents such as magnesium stearate. In the case of capsules, tablets andpills, the dosage forms may also comprise buffering agents. Tablets andpills can additionally be prepared with enteric coatings.

The polypeptides or conjugates may be admixed with adjuvants such aslactose, sucrose, starch powder, cellulose esters of alkanoic acids,stearic acid, talc, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodiumalginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tabletedor encapsulated for conventional administration. Alternatively, they maybe dissolved in saline, water, polyethylene glycol, propylene glycol,ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesameoil), tragacanth gum, and/or various buffers. Other adjuvants and modesof administration are well known in the pharmaceutical art. The carrieror diluent may include time delay material, such as glycerylmonostearate or glyceryl distearate alone or with a wax, or othermaterials well known in the art.

The pharmaceutical compositions may be subjected to conventionalpharmaceutical operations such as sterilization and/or may containconventional adjuvants such as preservatives, stabilizers, wettingagents, emulsifiers, buffers, fillers, etc., e.g. as disclosed elsewhereherein.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants such as wetting agents,sweeteners, flavoring agents and perfuming agents.

Formulations suitable for pulmonary administration are intended as partof the invention. Formulations suitable for use with a nebulizer, eitherjet or ultrasonic, will typically comprise the polypeptide or conjugatedissolved in water at a concentration of, e.g., about 0.01 to 25 mg ofconjugate per mL of solution, preferably about 0.1 to 10 mg/mL. Theformulation may also include a buffer and a simple sugar (e.g., forprotein stabilization and regulation of osmotic pressure), and/or humanserum albumin ranging in concentration from 0.1 to 10 mg/ml. Examples ofbuffers that may be used are sodium acetate, citrate and glycine.Preferably, the buffer will have a composition and molarity suitable toadjust the solution to a pH in the range of 3 to 9. Generally, buffermolarities of from 1 mM to 50 mM are suitable for this purpose. Examplesof sugars which can be utilized are lactose, maltose, mannitol,sorbitol, trehalose, and xylose, usually in amounts ranging from 1% to10% by weight of the formulation.

The nebulizer formulation may also contain a surfactant to reduce orprevent surface induced aggregation of the protein caused by atomizationof the solution in forming the aerosol. Various conventional surfactantscan be employed, such as polyoxyethylene fatty acid esters and alcohols,and polyoxyethylene sorbitan fatty acid esters. Amounts will generallyrange between 0.001% and 4% by weight of the formulation. An especiallypreferred surfactant for purposes of this invention is polyoxyethylenesorbitan monooleate.

Specific formulations and methods of generating suitable dispersions ofliquid particles of the invention are described in WO 94/20069, U.S.Pat. Nos. 5,915,378, 5,960,792, 5,957,124, 5,934,272, 5,915,378,5,855,564, 5,826,570 and 5,522,385 which are hereby incorporated byreference.

Formulations for use with a metered dose inhaler device will generallycomprise a finely divided powder. This powder may be produced bylyophilizing and then milling a liquid conjugate formulation and mayalso contain a stabilizer such as human serum albumin (HSA). Typically,more than 0.5% (w/w) HSA is added. Additionally, one or more sugars orsugar alcohols may be added to the preparation if necessary. Examplesinclude lactose maltose, mannitol, sorbitol, sorbitose, trehalose,xylitol, and xylose. The amount added to the formulation can range fromabout 0.01 to 200% (w/w), preferably from approximately 1 to 50%, of theconjugate present. Such formulations are then lyophilized and milled tothe desired particle size.

The properly sized particles are then suspended in a propellant with theaid of a surfactant. The propellant may be any conventional materialemployed for this purpose, such as a chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant. Thismixture is then loaded into the delivery device. An example of acommercially available metered dose inhaler suitable for use in thepresent invention is the Ventolin metered dose inhaler, manufactured byGlaxo Inc., Research Triangle Park, N.C., USA.

Formulations for powder inhalers will comprise a finely divided drypowder containing polypeptides or polypeptide conjugates and may alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, or mannitolin amounts which facilitate dispersal of the powder from the device,e.g., 50% to 90% by weight of the formulation. The particles of thepowder shall have aerodynamic properties in the lung corresponding toparticles with a density of about 1 g/cm² having a median diameter lessthan 10 micrometers, preferably between 0.5 and 5 micrometers, mostpreferably of between 1.5 and 3.5 micrometers. An example of a powderinhaler suitable for use in accordance with the teachings herein is theSpinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.,USA. The powders for these devices may be generated and/or delivered bymethods disclosed in U.S. Pat. Nos. 5,997,848, 5,993,783, 5,985,248,5,976,574, 5,922,354, 5,785,049 and 5,654,007.

Mechanical devices designed for pulmonary delivery of therapeuticproducts, include but are not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to those ofskill in the art. Specific examples of commercially available devicessuitable for the practice of this invention are the Ultravent nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo., USA; the Acorn IInebulizer, manufactured by Marquest Medical Products, Englewood, Colo.,USA; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C., USA; the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass., USA the “standing cloud”device of Nektar Therapeutics, Inc., San Carlos, Calif, USA; the AIRinhaler manufactured by Alkermes, Cambridge, Mass., USA; and the AERxpulmonary drug delivery system manufactured by Aradigm Corporation,Hayward, Calif, USA.

The present invention also provides kits including the polypeptides,conjugates, polynucleotides, expression vectors, cells, methods,compositions, and systems, and apparatuses of the invention. Kits of theinvention optionally comprise at least one of the following of theinvention: (1) an apparatus, system, system component, or apparatuscomponent as described herein; (2) at least one kit component comprisinga polypeptide or conjugate or polynucleotide of the invention; a plasmidexpression vector encoding a polypeptide of the invention; a cellexpressing a polypeptide of the invention; or a composition comprisingat least one of any such component; (3) instructions for practicing anymethod described herein, including a therapeutic or prophylactic method,instructions for using any component identified in (2) or anycomposition of any such component; and/or instructions for operating anyapparatus, system or component described herein; (4) a container forholding said at least one such component or composition, and (5)packaging materials.

In a further aspect, the present invention provides for the use of anyapparatus, component, composition, or kit described above and herein,for the practice of any method or assay described herein, and/or for theuse of any apparatus, component, composition, or kit to practice anyassay or method described herein.

Chemical Modifications, Conjugates, and Fusions

Any polypeptide of the invention may be present as part of a largerpolypeptide sequence, e.g. a fusion protein, such as occurs upon theaddition of one or more domains or subsequences for stabilization ordetection or purification of the polypeptide. A polypeptide purificationsubsequence may include, e.g., an epitope tag, a FLAG tag, apolyhistidine sequence, a GST fusion, or any otherdetection/purification subsequence or “tag” known in the art. Theseadditional domains or subsequences either have little or no effect onthe activity of the polypeptide of the invention, or can be removed bypost synthesis processing steps such as by treatment with a protease,inclusion of an intein, or the like.

The invention includes fusion proteins comprising a polypeptide of theinvention, e.g., as described herein, fused to an Ig molecule, e.g., ahuman IgG Fc (“fragment crystallizable,” or fragment complement binding)hinge, CH2 domain and CH3 domain, and nucleotide sequences encoding suchfusion protein. Fc is the portion of the antibody responsible forbinding to antibody receptors on cells and the C1q component ofcomplement. These fusion proteins and their encoding nucleic acids areuseful as prophylactic and/or therapeutic drugs or as diagnostic tools(see also, e.g., Challita-Eid, P. et al. (1998) J. Immunol160:3419-3426; Sturmhoefel, K. et al. (1999) Cancer Res 59:4964-4972).The invention also includes fusion proteins comprising a polypeptide ofthe invention, fused to an albumin molecule, such as human serum albumin(HSA), as described, for example, in U.S. Pat. No. 5,876,969, andnucleotide sequences encoding the fusion protein. The Ig and albuminfusion proteins may exhibit increased polypeptide serum half-life and/orfunctional in vivo half-life, reduced polypeptide antigenicity,increased polypeptide storage stability, or increasing bioavailability,e.g. increased AUC_(sc), and are thus may be useful as prophylacticand/or therapeutic drugs.

All of the polypeptides of the invention have an inherent ability totransduce across cellular membranes and affect therapeutic functionswithin cells. The invention therefore provides for the use of thepolypeptides of the invention to enhance the cell permeability ortransducibility of any other molecule. The invention further providesfor the use of any fragment or subfragment of the polypeptides of theinvention to enhance the cell permeability of any other molecule, suchfragments or subfragments being of about 5 amino acids in length, ofabout 10 amino acids in length, such as 15 amino acids in length, e.g.about 20 amino acids in length, of about 25 amino acids in length, ofabout 30 amino acids in length, such as 35 amino acids in length, ofabout 35-50 amino acids in length, of about 50-100 amino acids inlength, such as 75 amino acids in length, e.g. 100-125 amino acids inlength.

Any polypeptide of the invention may also comprise one or more modifiedamino acid. The modified amino acid may be, e.g., a glycosylated aminoacid, a PEGylated amino acid, a famesylated amino acid, an acetylatedamino acid, a biotinylated amino acid, an amino acid conjugated to alipid moiety, or an amino acid conjugated to an organic derivatizingagent. The presence of modified amino acids may be advantageous in, forexample, (a) increasing polypeptide serum half-life and/or functional invivo half-life, (b) reducing polypeptide antigenicity, (c) increasingpolypeptide storage stability, or (d) increasing bioavailability, e.g.increasing the AUC_(sc). Amino acid(s) are modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation at N-X-S/T motifs during expression inmammalian cells) or modified by synthetic means.

The term “conjugate” (or interchangeably “polypeptide conjugate” or“conjugated polypeptide”) is intended to indicate a heterogeneous (inthe sense of composite) molecule formed by the covalent attachment ofone or more polypeptides of the invention to one or more non-polypeptidemoieties. The term “covalent attachment” means that the polypeptide andthe non-polypeptide moiety are either directly covalently joined to oneanother, or else are indirectly covalently joined to one another throughan intervening moiety or moieties, such as a bridge, spacer, or linkagemoiety or moieties. Preferably, a conjugated polypeptide is soluble atrelevant concentrations and conditions, i.e. soluble in physiologicalfluids such as blood. Examples of conjugated polypeptides of theinvention include glycosylated and/or PEGylated polypeptides. The term“non-conjugated polypeptide” may be used to refer to the polypeptidepart of the conjugated polypeptide.

The term “non-polypeptide moiety” is intended to mean a molecule that iscapable of conjugating to an attachment group of the polypeptide.Preferred examples of non-polypeptide moieties include polymermolecules, sugar moieties, lipophilic compounds, or organic derivatizingagents, in particular polymer molecules or sugar moieties. It will beunderstood that the non-polypeptide moiety is linked to the polypeptidethrough an attachment group of the polypeptide. Except where the numberof non-polypeptide moieties, such as polymer molecule(s), attached tothe polypeptide is expressly indicated, every reference to “anon-polypeptide moiety” attached to the polypeptide or otherwise used inthe present invention shall be a reference to one or morenon-polypeptide moieties attached to the polypeptide.

The term “polymer molecule” is defined as a molecule formed by covalentlinkage of two or more monomers, wherein none of the monomers is anamino acid residue. The term “polymer” may be used interchangeably withthe term “polymer molecule”.

The term “sugar moiety” is intended to indicate a carbohydrate moleculeattached by in vivo or in vitro glycosylation, such as N- orO-glycosylation. An “N-glycosylation site” has the sequence N-X-S/T/C,wherein X is any amino acid residue except proline, N is asparagine andS/T/C is either serine, threonine or cysteine, preferably serine orthreonine, and most preferably threonine. An “O-glycosylation site”comprises the OH-group of a serine or threonine residue.

The term “attachment group” is intended to indicate an amino acidresidue group capable of coupling to the relevant non-polypeptide moietysuch as a polymer molecule or a sugar moiety.

For in vivo N-glycosylation, the term “attachment group” is used in anunconventional way to indicate the amino acid residues constituting anN-glycosylation site (with the sequence N-X-S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine). Although the asparagine residue of theN-glycosylation site is the one to which the sugar moiety is attachedduring glycosylation, such attachment cannot be achieved unless theother amino acid residues of the N-glycosylation site is present.Accordingly, when the non-polypeptide moiety is a sugar moiety and theconjugation is to be achieved by N-glycosylation, the term “amino acidresidue comprising an attachment group for the non-polypeptide moiety”as used in connection with alterations of the amino acid sequence of thepolypeptide of the invention is to be understood as one, two or all ofthe amino acid residues constituting an N-glycosylation site is/are tobe altered in such a manner that either a functional N-glycosylationsite is introduced into the amino acid sequence, removed from saidsequence, or a functional N-glycosylation site is retained in the aminoacid sequence (e.g. by substituting a serine residue, which alreadyconstitutes part of an N-glycosylation site, with a threonine residueand vice versa).

The term “introduce” (i.e., an “introduced” amino acid residue,“introduction” of an amino acid residue) is primarily intended to meansubstitution of an existing amino acid residue for another amino acidresidue, but may also mean insertion of an additional amino acidresidue.

The term “remove” (i.e., a “removed” amino acid residue, “removal” of anamino acid residue) is primarily intended to mean substitution of theamino acid residue to be removed for another amino acid residue, but mayalso mean deletion (without substitution) of the amino acid residue tobe removed.

The term “amino acid residue comprising an attachment group for thenon-polypeptide moiety” is intended to indicate that the amino acidresidue is one to which the non-polypeptide moiety binds (in the case ofan introduced amino acid residue) or would have bound (in the case of aremoved amino acid residue).

The term “functional in vivo half-life” is used in its normal meaning,i.e. the time at which 50% of the biological activity of the polypeptideis still present in the body/target organ, or the time at which theactivity of the polypeptide is 50% of the initial value. The functionalin vivo half-life may be determined in an experimental animal, such asrat, mouse, rabbit, dog or monkey. Preferably, the functional in vivohalf-life is determined in a non-human primate, such as a monkey.Furthermore, the functional in vivo half-life may be determined for asample that has been administered intravenously or subcutaneously.

As an alternative to determining functional in vivo half-life, “serumhalf-life” may be determined, i.e. the time at which 50% of thepolypeptide circulates in the plasma or bloodstream prior to beingcleared. Determination of serum half-life is often more simple thandetermining the functional in vivo half-life and the magnitude of serumhalf-life is usually a good indication of the magnitude of functional invivo half-life. Alternatively terms to serum half-life include “plasmahalf-life”, “circulating half-life”, “serum clearance”, “plasmaclearance” and “clearance half-life”.

Polynucleotides and Methods of Mutagenesis

The invention includes nucleic acids and polynucleotides that encode thepolypeptides of the invention. The invention includes compositionsproduced by digesting one or more of any of the polynucleotides of theinvention with a restriction endonuclease, an RNAse, or a DNAse (e.g.,as is performed in certain of the recombination formats elsewhere in thespecification); and compositions produced by fragmenting or shearing oneor more polynucleotides of the invention by mechanical means (e.g.,sonication, vortexing, and the like), which can also be used to providesubstrates for recombination in the methods described herein. Theinvention also provides compositions produced by cleaving at least oneof any of the polynucleotides of the invention. The cleaving maycomprise mechanical, chemical, or enzymatic cleavage, and the enzymaticcleavage may comprise cleavage with a restriction endonuclease, anRNAse, or a DNAse.

Also included in the invention are compositions produced by a processcomprising incubating one or more of the fragmented polynucleotides ofthe invention in the presence of ribonucleotide or deoxyribonucleotidetriphosphates and a nucleic acid polymerase. This resulting compositionforms a recombination mixture for many of the recombination formatsnoted above. The nucleic acid polymerase may be an RNA polymerase, a DNApolymerase, or an RNA-directed DNA polymerase (e.g., a “reversetranscriptase”); the polymerase can be, e.g., a thermostable DNApolymerase (e.g., VENT, TAQ, or the like).

Similarly, compositions comprising sets of oligonucleotidescorresponding to more than one nucleic acids of the invention are usefulas recombination substrates and are a feature of the invention. Forconvenience, these fragmented, sheared, or oligonucleotide synthesizedmixtures are referred to as fragmented nucleic acid sets.

The invention also provides an isolated or recombinant nucleic acidencoding a polypeptide produced by mutating or recombining at least onepolynucleotide of the invention.

Polynucleotides, oligonucleotides, and nucleic acid fragments of theinvention can be prepared by standard solid-phase methods, according toknown synthetic methods. Typically, fragments of up to about 100 basesare individually synthesized, then joined (e.g., by enzymatic orchemical ligation methods, or polymerase mediated recombination methods)to form essentially any desired continuous sequence. For example, thepolynucleotides and oligonucleotides of the invention can be prepared bychemical synthesis using, e.g., classical phosphoramidite methoddescribed by, e.g., Beaucage et al. (1981) Tetrahedron Letters22:1859-69, or the method described by Matthes et al. (1984) EMBO J3:801-05, e.g., as is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned into appropriate vectors.

In addition, essentially any polynucleotide can be custom ordered fromany of a variety of commercial sources, such as Operon Technologies Inc.(Alameda, Calif.) and many others. Similarly, peptides and antibodiescan be custom ordered from any of a variety of sources, e.g., CeltekPeptides (Nashville, Tenn.); Washington Biotechnology, Inc. (BaltimoreMd.); Global Peptide Services (Ft. Collin Colo.), and many others.

Certain polynucleotides of the invention may also be obtained byscreening cDNA libraries (e.g., libraries generated by recombininghomologous nucleic acids as in typical recursive sequence recombinationmethods) using oligonucleotide probes that can hybridize to orPCR-amplify polynucleotides which encode OAS polypeptides and fragmentsof those polypeptides. Procedures for screening and isolating cDNAclones are well-known to those of skill in the art. Such techniques aredescribed in, e.g., Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymol. Vol. 152, Acad. Press, Inc., San Diego,Calif. (“Berger”); J. Sambrook and D. W. Russell, Molecular Cloning: ALaboratory Manual, Third Edition. Cold Spring Harbor Press, Cold SpringHarbor, N.Y., (“Sambrook”); and F.M. Ausubel et al. (1987-2005) CurrentProtocols in Molecular Biology. Wiley Interscience, New York, N.Y.(“Ausubel”). Some polynucleotides of the invention can be obtained byaltering a naturally occurring sequence, e.g., by mutagenesis, recursivesequence recombination (e.g., shuffling), or oligonucleotiderecombination. In other cases, such polynucleotides can be made insilico or through oligonucleotide recombination methods as described inthe references cited herein.

As described in more detail herein, the polynucleotides of the inventioninclude polynucleotides that encode polypeptides of the invention,polynucleotide sequences complementary to these polynucleotidesequences, and polynucleotides that hybridize under at least stringentconditions to the sequences defined herein. A coding sequence refers toa polynucleotide sequence encoding a particular polypeptide or domain,region, or fragment of said polypeptide. The polynucleotides of theinvention may be in the form of RNA or in the form of DNA, and includemRNA, cRNA, synthetic RNA and DNA, and cDNA. The polynucleotides may bedouble-stranded or single-stranded, and if single-stranded, can be thecoding strand or the non-coding (anti-sense, complementary) strand. Thepolynucleotides of the invention include the coding sequence of apolypeptide of the invention (i) in isolation, (ii) in combination withone or more additional coding sequences, so as to encode, e.g., a fusionprotein, a pre-protein, a prepro-protein, or the like, (iii) incombination with non-coding sequences, such as introns, controlelements, such as a promoter (e.g., naturally occurring or recombinantor shuffled promoter), a terminator element, or 5′ and/or 3′untranslated regions effective for expression of the coding sequence ina suitable host, and/or (iv) in a vector, cell, or host environment inwhich the coding sequence is a heterologous gene.

Polynucleotides of the invention can also be found in combination withtypical compositional formulations of nucleic acids, including in thepresence of carriers, buffers, adjuvants, excipients, and the like, asare known to those of ordinary skill in the art. Polynucleotidefragments typically comprise at least about 200 nucleotide bases, suchas at least about 250, 300, 350, 400, 450, 460, 470, or more bases. Thenucleotide fragments of polynucleotides of the invention may hybridizeunder highly stringent conditions to a polynucleotide sequence describedherein and/or encode amino acid sequences having at least one of theproperties of polypeptides of the invention described herein.

The polynucleotides of the invention have a variety of uses in, forexample, recombinant production (i.e., expression) of the polypeptidesof the invention typically through expression of a plasmid expressionvector comprising a sequence encoding the polypeptide or fragmentthereof; as therapeutics; as prophylactics; as diagnostic tools; asimmunogens; as adjuvants; as diagnostic probes for the presence ofcomplementary or partially complementary nucleic acids (including fordetection of a wild-type oligoadenylate synthetase nucleic acid), assubstrates for further reactions, e.g., recursive sequence recombinationreactions or mutation reactions to produce new and/or improved variants,and the like.

Expression Vectors, Methods of Manufacturing, Gene Therapy

Recombinant methods for producing and isolating polypeptides of theinvention are described herein. In addition to recombinant production,the polypeptides may be produced by direct peptide synthesis usingsolid-phase techniques (see, e.g., Stewart et al. (1969) Solid-PhasePeptide Synthesis, WH Freeman Co, San Francisco; Merrifield J. (1963) JAm Chem Soc 85:2149-2154). Peptide synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Foster City, Calif.) in accordance with the instructions providedby the manufacturer. For example, subsequences may be chemicallysynthesized separately and combined using chemical methods to providefull-length polypeptides or fragments thereof. Alternatively, suchsequences may be ordered from any number of companies which specializein production of polypeptides. Most commonly, polypeptides of theinvention may be produced by expressing coding nucleic acids andrecovering polypeptides, e.g., as described below.

Methods for producing the polypeptides of the invention are alsoincluded. One such method comprises introducing into a population ofcells any nucleic acid of the invention, which is operatively linked toa regulatory sequence effective to produce the encoded polypeptide,culturing the cells in a culture medium to express the polypeptide, andisolating the polypeptide from the cells or from the culture medium. Anamount of nucleic acid sufficient to facilitate uptake by the cells(transfection) and/or expression of the polypeptide is utilized. Thenucleic acid is introduced into such cells by any delivery method as isknown in the art, including, e.g., injection, gene gun, passive uptake,etc. As one skilled in the art will recognize, the nucleic acid may bepart of a vector, such as a recombinant expression vector, including aDNA plasmid vector, or any vector as known in the art. The nucleic acidor vector comprising a nucleic acid of the invention may be prepared andformulated by standard recombinant DNA technologies and isolationmethods as known in the art. Such a nucleic acid or expression vectormay be introduced into a population of cells of a mammal in vivo, orselected cells of the mammal (e.g., tumor cells) may be removed from themammal and the nucleic acid expression vector introduced ex vivo intothe population of such cells in an amount sufficient such that uptakeand expression of the encoded polypeptide results. Or, a nucleic acid orvector comprising a nucleic acid of the invention is produced usingcultured cells in vitro. In one aspect, the method of producing apolypeptide of the invention comprises introducing into a population ofcells a recombinant expression vector comprising any nucleic acid of theinvention described herein in an amount and formula such that uptake ofthe vector and expression of the encoded polypeptide will result;administering the expression vector into a mammal by anyintroduction/delivery format described herein; and isolating thepolypeptide from the mammal or from a byproduct of the mammal.

The invention provides isolated or recombinant nucleic acids (alsoreferred to herein as polynucleotides), collectively referred to as“nucleic acids (or polynucleotides) of the invention”, which encodepolypeptides of the invention. The polynucleotides of the invention areuseful in a variety of applications. As discussed above, thepolynucleotides are useful in producing polypeptides of the invention.In addition, polynucleotides of the invention can be incorporated intoexpression vectors useful for gene therapy, DNA vaccination, andimmunotherapy, as described elsewhere in this application.

Any of the polynucleotides of the invention (which includes thosedescribed above) may encode a fusion protein comprising at least oneadditional amino acid sequence, such as, for example, asecretion/localization sequence, a sequence useful for solubilization orimmobilization (e.g., for cell surface display) of the polypeptide, asequence useful for detection and/or purification of the polypeptide(e.g., a polypeptide purification subsequence, such as an epitope tag, apolyhistidine sequence, and the like). In another aspect, the inventionprovides cells comprising one or more of the polynucleotides of theinvention. Such cells may express one or more polypeptides encoded bythe polynucleotides of the invention.

The invention also provides vectors comprising any of thepolynucleotides of the invention. Such vectors may comprise a plasmid, acosmid, a phage, a virus, or a fragment of a virus. Such vectors maycomprise an expression vector, and, if desired, the nucleic acid isoperably linked to a promoter, including those discussed herein andbelow. Furthermore, in another aspect, the invention providescompositions comprising an excipient or carrier and at least one of anyof the polynucleotides of the invention, or vectors, cells, or hostcomprising such nucleic acids. Such composition may be pharmaceuticalcompositions, and the excipient or carrier may be a pharmaceuticallyacceptable excipient or carrier.

The invention also includes compositions comprising two or more nucleicacids of the invention, or fragments thereof (e.g., as substrates forrecombination). The composition can comprise a library of recombinantnucleic acids, where the library contains at least 2, at least 3, atleast 5, at least 10, at least 20, at least 50, or at least 100 or morenucleic acids described above. The nucleic acids are optionally clonedinto expression vectors, providing expression libraries.

The polynucleotides of the invention and fragments thereof, as well asvectors comprising such polynucleotides, may be employed for therapeuticor prophylactic uses in combination with a suitable carrier, such as apharmaceutical carrier. Such compositions comprise a therapeuticallyand/or prophylactically effective amount of the compound, and apharmaceutically acceptable carrier or excipient. Such a carrier orexcipient includes, but is not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration. Methods ofadministering nucleic acids, polypeptides, and proteins are well knownin the art.

General texts that describe molecular biological techniques usefulherein, including the use of vectors, promoters and many other relevanttopics, include Berger, supra; Sambrook (1989), supra, and Ausubel,supra. Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods, including the polymerase chainreaction (PCR) the ligase chain reaction (LCR), Q beta-replicaseamplification and other RNA polymerase mediated techniques (e.g.,NASBA), e.g., for the production of the homologous nucleic acids of theinvention are found in Berger, Sambrook, and Ausubel, all supra, as wellas Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols: A Guideto Methods and Applications (Innis et al., eds.) Academic Press Inc. SanDiego, Calif. (1990) (“Innis”); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3:81-94; (Kwoh et al. (1989)Proc Natl Acad Sci USA 86:1173-1177; Guatelli et al. (1990) Proc NatlAcad Sci USA 87:1874-1878; Lomeli et al. (1989) J Clin Chem35:1826-1831; Landegren et al. (1988) Science 241:1077-1080; Van Brunt(1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4:560-569;Barringer et al. (1990) Gene 89:117-122, and Sooknanan and Malek (1995)Biotechnology 13:563-564. Improved methods of cloning in vitro amplifiednucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039.Improved methods of amplifying large nucleic acids by PCR are summarizedin Cheng et al. (1994) Nature 369:684-685 and the references therein, inwhich PCR amplicons of up to 40 kilobases (kb) are generated. One ofskill will appreciate that essentially any RNA can be converted into adouble stranded DNA suitable for restriction digestion, PCR expansionand sequencing using reverse transcriptase and a polymerase. SeeAusubel, Sambrook and Berger, all supra.

In mammalian host cells, a number of expression systems, such asviral-based systems, may be utilized. In cases where an adenovirus isused as an expression vector, a coding sequence is optionally ligatedinto an adenovirus transcription/translation complex consisting of thelate promoter and tripartite leader sequence. Insertion in anonessential E1 or E3 region of the viral genome results in a viablevirus capable of expressing a polypeptide of the invention in infectedhost cells (Logan and Shenk (1984) Proc Natl Acad Sci USA 81:3655-3659).In addition, transcription enhancers, such as the rous sarcoma virus(RSV) enhancer, are used to increase expression in mammalian host cells.Host cells, media, expression systems, and methods of production includethose known for cloning and expression of various mammalian proteins.The efficiency of expression can be enhanced by the inclusion ofenhancers appropriate to the cell system in use (see, e.g., Scharf D. etal. (1994) Results Probl Cell Differ 20:125-62; and Bittner et al.(1987) Methods in Enzymol 153:516-544).

Specific initiation signals can aid in efficient translation of apolynucleotide coding sequence of the invention and/or fragmentsthereof. These signals can include, e.g., the ATG initiation codon andadjacent sequences. In cases where a coding sequence, its initiationcodon and upstream sequences are inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only coding sequence (e.g., a matureprotein coding sequence), or a portion thereof, is inserted, exogenousnucleic acid transcriptional control signals including the ATGinitiation codon must be provided. Furthermore, the initiation codonmust be in the correct reading frame to ensure transcription of theentire insert. Exogenous transcriptional elements and initiation codonscan be of various origins, both natural and synthetic.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-Dextran mediated transfection,electroporation, gene or vaccine gun, injection, or other commontechniques (see, e.g., Davis, L., Dibner, M., and Battey, I. (1986)Basic Methods in Molecular Biology) for in vivo, ex vivo or in vitromethods.

As noted, many references are available for the culture and productionof many cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin. See, e.g., Sambrook, Ausubel, andBerger (all supra), as well as Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein; Doyle and Griffiths (1997) Mammalian CellCulture: Essential Techniques John Wiley and Sons, New York; Humason(1979) Animal Tissue Techniques, fourth edition W.H. Freeman andCompany; and Ricciardelli et al. (1989) In vitro Cell Dev Biol25:1016-1024. For plant cell culture and regeneration see, e.g., Payneet al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley& Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Plant Molecular Biology(1993) R. R. D. Croy (ed.) Bios Scientific Publishers, Oxford, U.K. ISBN0 12 198370 6. Cell culture media in general are set forth in Atlas andParks (eds.) The Handbook of Microbiological Media (1993) CRC Press,Boca Raton, Fla. Additional information for cell culture is found inavailable commercial literature such as the Life Science Research CellCulture Catalogue from Sigma-Aldrich, Inc (St Louis, Mo.)(“Sigma-LSRCCC”) and, e.g., the Plant Culture Catalogue and supplementalso from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-PCCS”).

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by any of a number of methods well known inthe art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems noted herein),hydroxylapatite chromatography, and lectin chromatography. Proteinrefolding steps can be used, as desired, in completing configuration ofthe mature protein or fragments thereof. Finally, high performanceliquid chromatography (HPLC) can be employed in the final purificationsteps. In addition to the references noted, supra, a variety ofpurification methods are well known in the art, including, e.g., thoseset forth in Sandana (1997) Bioseparation of Proteins, Academic Press,Inc.; Bollag et al. (1996) Protein Methods, 2.sup.nd Edition Wiley-Liss,New York; Walker (1996) The Protein Protocols Handbook Humana Press, NewJersey; Harris and Angal (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, England; Harris andAngal Protein Purification Methods: A Practical Approach IRL Press atOxford, Oxford, England; Scopes (1993) Protein Purification: Principlesand Practice 3.sup.rd Edition Springer Verlag, New York; Janson andRyden (1998) Protein Purification: Principles, High Resolution Methodsand Applications, Second Edition Wiley-VCH, New York; and Walker (1998)Protein Protocols on CD-ROM Humana Press, New Jersey.

A number of viral vectors suitable for organismal in vivo transductionand expression are known. Such vectors include retroviral vectors (see,e.g., Miller, Curr Top Microbiol Immunol (1992) 158:1-24; Salmons andGunzburg (1993) Human Gene Therapy 4:129-141; Miller et al. (1994)Methods in Enzymology 217:581-599) and adeno-associated vectors(reviewed in Carter (1992) Curr Opinion Biotech 3:533-539; Muzcyzka(1992) Curr Top Microbiol Immunol. 158:97-129). Other viral vectors thatare used include adenoviral vectors, herpes viral vectors and Sindbisviral vectors, as generally described in, e.g., Jolly (1994) Cancer GeneTherapy 1:51-64; Latchman (1994) Molec Biotechnol 2:179-195; andJohanning et al. (1995) Nucl Acids Res 23:1495-1501.

In one aspect, a pox virus vector can be used. The pox viral vector istransfected with a polynucleotide sequence encoding a polypeptide of theinvention and is useful in prophylactic, therapeutic and diagnosticapplications where enhancement of an immune response, such as e.g.,increased or improved T cell proliferation is desired. See viral vectorsdiscussed in, e.g., Berencsi et al., J Infect Dis (2001)183(8):1171-9;Rosenwirth et al., Vaccine 2001 February 8;19(13-14):1661-70; Kittlesenet al., J Immunol (2000) 164(8):4204-11; Brown et al. Gene Ther 20007(19):1680-9; Kanesa-thasan et al., Vaccine (2000) 19(4-5):483-91; Sten(2000) Drug 60(2):249-71. Compositions comprising such vectors and anacceptable excipient are also a feature of the invention.

Gene therapy and genetic vaccines provide methods for combating chronicinfectious diseases (e.g., HIV infection, viral hepatitis), as well asnon-infectious diseases including cancer and some forms of congenitaldefects such as enzyme deficiencies, and such methods can be employedwith polynucleotides of the invention, including, e.g., vectors andcells comprising such polynucleotides. Several approaches forintroducing nucleic acids and vectors into cells in vivo, ex vivo and invitro have been used and can be employed with polynucleotides of theinvention, and vectors comprising such polynucleotides. These approachesinclude liposome based gene delivery (Debs and Zhu (1993) WO 93/24640and U.S. Pat. No. 5,641,662; Mannino and Gould-Fogerite (1988)BioTechniques 6(7):682-691; Rose, U.S. Pat. No. 5,279,833; Brigham(1991) WO 91/06309; and Feigner et al. (1987) Proc Natl Acad Sci USA84:7413-7414; Brigham et al. (1989) Am J Med Sci 298:278-281; Nabel etal. (1990) Science 249:1285-1288; Hazinski et al. (1991) Am J Resp CellMolec Biol 4:206-209; and Wang and Huang (1987) Proc Natl Acad Sci USA84:7851-7855); adenoviral vector mediated gene delivery, e.g., to treatcancer (see, e.g., Chen et al. (1994) Proc Nati Acad Sci USA91:3054-3057; Tong et al. (1996) Gynecol Oncol 61:175-179; Clayman etal. (1995) Cancer Res. 5:1-6; O'Malley et al. (1995) Cancer Res55:1080-1085; Hwang et al. (1995) Am J Respir Cell Mol Biol 13:7-16;Haddada et al. (1995) Curr Top Microbiol Immunol. 1995 (Pt. 3):297-306;Addison et al. (1995) Proc Nati Acad Sci USA 92:8522-8526; Colak et al.(1995) Brain Res 691:76-82; Crystal (1995) Science 270:404-410; Elshamiet al. (1996) Human Gene Ther 7:141-148; Vincent et al. (1996) JNeurosurg 85:648-654), and many others. Replication-defective retroviralvectors harboring therapeutic polynucleotide sequence as part of theretroviral genome have also been used, particularly with regard tosimple MuLV vectors. See, e.g., Miller et al. (1990) Mol Cell Biol10:4239 (1990); Kolberg (1992) J NIH Res 4:43, and Cornetta et al.(1991) Hum Gene Ther 2:215). Nucleic acid transport coupled toligand-specific, cation-based transport systems (Wu and Wu (1988) J BiolChem, 263:14621-14624) has also been used. Naked DNA expression vectorshave also been described (Nabel et al. (1990), supra); Wolff et al.(1990) Science, 247:1465-1468). In general, these approaches can beadapted to the invention by incorporating nucleic acids encoding thepolypeptides of the invention into the appropriate vectors.

General texts which describe gene therapy protocols, which can beadapted to the present invention by introducing the nucleic acids of theinvention into patients, include, e.g., Robbins (1996) Gene TherapyProtocols, Humana Press, New Jersey, and Joyner (1993) Gene Targeting: APractical Approach, IRL Press, Oxford, England.

Antiviral Treatments

The polynucleotides and polypeptides of the invention may be usedtherapeutically or prophylactically to treat or prevent virus infection.Exemplary viruses include, but are not limited to, viruses of theFlaviviridae family, such as, for example, Hepatitis C Virus, YellowFever Virus, West Nile Virus, Japanese Encephalitis Virus, Dengue Virus,and Bovine Viral Diarrhea Virus; viruses of the Hepadnaviridae family,such as, for example, Hepatitis B Virus; viruses of the Picornaviridaefamily, such as, for example, Encephalomyocarditis Virus, HumanRhinovirus, and Hepatitis A Virus; viruses of the Retroviridae family,such as, for example, Human Immunodeficiency Virus, SimianImmunodeficiency Virus, Human T-Lymphotropic Virus, and Rous SarcomaVirus; viruses of the Coronaviridae family, such as, for example, SARScoronavirus; viruses of the Rhabdoviridae family, such as, for example,Rabies Virus and Vesicular Stomatitis Virus, viruses of theParamyxoviridae family, such as, for example, Respiratory SyncytialVirus and Parainfluenza Virus, viruses of the Papillomaviridae family,such as, for example, Human Papillomavirus, and viruses of theHerpesviridae family, such as, for example, Herpes Simplex Virus.

It is another object of the invention to provide conjugates, suchconjugates comprising one or more non-polypeptide moiety linked to apolypeptide of the invention, which conjugate exhibits an antiviralproperty, and which optionally exhibits other desirable properties, suchas increased serum half-life and/or functional in vivo half-life, and/ordecreased antigenicity, compared to the non-conjugated polypeptide. Somesuch conjugates may exhibit enhanced efficacy in clearing a virus fromcells infected with the virus, compared to a reference oligoadenylatesynthetase. Some such conjugates may further have reduced toxicitycompared to a reference oligoadenylate synthetase.

It is another object of the invention to provide a method of inhibitingviral replication in virus-infected cells, the method comprisingadministering to the virus-infected cells a polypeptide or conjugate ofthe invention in an amount effective to inhibit viral replication insaid cells. The invention also provides a method of reducing the numberof copies of a virus in virus-infected cells, comprising administeringto the virus-infected cells a polypeptide or conjugate of the inventionin an amount effective to reduce the number of copies of the virus insaid cells. The cells may be in culture or otherwise isolated from amammal (i.e., in vitro or ex vivo), or may be in vivo, e.g., in asubject, in a mammal, in a primate, or in man.

Anticancer and Inflammation Treatments

It has been demonstrated that the polypeptides of the invention cancause certain cell types and cell lines to undergo apoptosis or toaffect growth retardation of said cell lines or cell types. Such celllines or cell types include in an exemplary embodiment those derivedfrom the prostate and breast.

The invention provides a method of inhibiting proliferation of a cellpopulation, comprising contacting the cell population with a polypeptideof the invention in an amount effective to decrease proliferation of thecell population. The cell population may be in culture or otherwiseisolated from a mammal (i.e., in vitro or ex vivo), or may be in vivo,e.g., in a subject, in a mammal, a primate, or man.

The invention provides for treating cancers and neoplastic diseasesusing the polypeptides and polynucleotides of the invention. Exemplarycancers and neoplastic diseases include but are not limited to:adrenocortical carcinoma, AIDS related cancers, such as for example,Kaposi's sarcoma, AIDS-related lymphoma, anal cancer, astrocytoma, basalcell carcinoma, bile duct cancers, such as for example those of anextrahepatic nature, bladder cancer, bone cancers, such as for exampleosteosarcomas and malignant fibrous histiocytomas, brain stem glioma,brain tumors, such as for example gliomas, astrocytomas, malignantgliomas, ependymomas, medulloblastomas, and neuroblastomas,supratentorial primitive neuroectodermal tumor, visual pathway andhypothalamic glioma, breast cancer, bronchial adenoma, Burkitt'slymphoma, carcinoid tumors, central nervous system lymphoma, cervicalcancer, leukemias, such as for example, hairy cell leukemia, acutelymphoblastic leukemia, acute myeloid leukemia, chronic lymphocyticleukemia and chronic myelogenous leukemia, chronic myeloproliferativedisorders, colorectal cancer, cutaneous T-cell lymphoma, endometrialcancer, esophageal cancer, Ewing's family of tumors, extracranial germcell tumor, extragonadal germ cell tumor, eye cancers, such as forexample, intraocular melanoma and retinoblastoma, gallbladder cancer,stomach cancer, gestational trophoblastic tumor, head and neck cancer,hepatocellular carcinoma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma,primary CNS lymphoma, nasopharyngeal cancer, islet cell carcinoma,kidney (renal cell) cancer, laryngeal cancer, lip and oral cancer, livercancer, lung cancer, such as for example non-small cell and small celllung cancers, Waldenstrom's macroglobulinemia, Merkel cell carcinoma,mesothelioma, metastatic squamous neck cancer, multiple endocrineneoplasia, multiple myeloma, plasma cell neoplasm, mycosis fungoides,myelodysplastic syndromes, myeloproliferative diseases, nasal cavity andparanasal sinus cancer, ovarian cancer, such as germ cell andepithelial, low-malignant potential ovarian tumor, pancreatic cancer,parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor,pleuropulmonary blastoma, prostate cancer, rhabdomyosarcoma, salivarygland cancer, sarcomas, Sezary syndrome, skin cancer, such as forexample melanoma and squamous cell carcinoma, testicular cancer,thymoma, thymic carcinoma, thyroid cancer, transitional cell cancer,trophoblastic tumor, urethral cancer, uterine cancer, vaginal cancer,vulvar cancer, and Wilms' tumor.

The invention further provides for treating autoimmune diseases andinflammation using the polypeptides and polynucleotides of theinvention, said autoimmune and inflammatory diseases include but are notlimited to: asthma, Crohn's disease, Guillain-Barre syndrome, multiplesclerosis, myasthenia gravis, optic neuritis, psoriasis, rheumatoidarthritis, Grave's disease, Hashimoto's (thyroiditis) disease, Ord'sthyroiditis, diabetes, diabetes mellitus, Reiter's syndrome, autoimmunehepatitis, primary biliary cirrhosis, liver cirrhosis, liver fibrosis,antiphospholipd antibody syndrome, opsoclonus myoclonus syndrome,temporal arteritis, acute disseminated encephalomyelitis, Goodpasture'ssyndrome, Wegener's granulomatosis, coeliac disease, pemphigus,polyarthritis, warm autoimmune hemolytic anemia, Takayasu's arteritis,coronary artery disease, endometriosis, interstitial cystitis,neuromyotonia, scleroderma, vitiligo, vulvodynia, Chagas' disease,sarcoidosis, chronic fatigue syndrome, acute respiratory distresssyndrome, tendonitis, bursitis, polymyalgia rheumatica, inflammatorybowel disease, chronic obstructive pulmonary disease, allergic rhinitis,cardiovascular disease, chronic cholecystitis, bronchiectasis,pneumoconiosis, such as for example, silicosis, osteoarthritis,atherosclerosis, dysautonomia, ankylosing spondylitis, acute anterioruvelitis, systemic lupus erythematosus, insulin-dependent diabetesmellitus, pemphigus vulgaris, experimental allergic encephalomyelitis,experimental autoimmune uveorenitis, mixed connective tissue disease,Sjorgen's syndrome, autoimmune hemolytic anemia, autoimmunethrombocytopenic purpura, acute rheumatic fever, mixed essentialcryoglobulinemia, juvenile rheumatoid arthritis, degenerative jointdisease, ankylosing spondylitis, psoriatic arthritis, neuralgia,synoviitis, glomerulonephritis, vasculitis, inflammations that occur assequellae to influenza, the common cold and other viral infections,gout, contact dermatitis, low back and neck pain, dysmenorrhea,headache, toothache, sprains, strains, myositis, burns, injuries, andpain and inflammation that follow surgical and dental procedures in asubject.

Cell Growth and Tissue Regeneration Treatments

The polypeptides of the invention have been shown to stimulate amitogenic, cell growth-promoting program in specific cell types and celllines, such as for example, Huh7 hepatoma cells and MRC5 fetal lungfibroblast cells. This mitogenic program is identified using expressionmicroarray analysis and cell viability assays of cells and cell linestreated with the polypeptides of the invention. The invention providesfor uses of the polypeptides of the invention to stimulate cell growthand tissue regeneration in vitro, in vivo, and ex vivo using tissues andcells derived from subjects or mammals.

Derivatives of the Polypeptides of the Invention

The invention provides for polypeptides that differ from any of thepolypeptides of FIGS. 1-5 by 1 to 34 amino acids, such differences mayinclude substitutions, insertions, deletions, the incorporation ofmodified amino acids or amino acid derivatives, and the addition ordeletion of amino acids from the C-terminus or N-terminus of thepolypeptides. One or more amino acid substitutions may be made to thepolypeptides of the invention according to, for example, a substitutiongroup (such as, a conservative substitution group), such as one setforth below. Alternatively, or in addition, one or more amino acidsubstitutions may made in the polypeptides which introduces or removesan amino acid residue comprising an attachment group for anon-polypeptide moiety. Examples include introduction of one or moreN-glycosylation site(s), introduction of one or more cysteine residue(s)or lysine residue(s), removal of one or more N-glycosylation site(s),and/or or removal of one or more lysine(s) or histidine(s). Some suchpolypeptides exhibit an oligoadenylate synthetase activity. Conservativesubstitutions groups include: Group 1, Alanine (A) Glycine (G) Serine(S) Threonine (T), Group 2, Aspartic acid (D) Glutamic acid (E), Group3, Asparagine (N) Glutamine (Q), Group 4, Arginine (R) Lysine (K)Histidine (H), Group 5, Isoleucine (I) Leucine (L) Methionine (M) Valine(V), and Group 6, Phenylalanine (F) Tyrosine (Y) Tryptophan (W). Othersubstitution groups of amino acids can be envisioned. For example, aminoacids can be grouped by similar function or chemical structure orcomposition (e.g., acidic, basic, aliphatic, aromatic,sulfur-containing). For example, an Aliphatic grouping may comprise:Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I). Othergroups containing amino acids that are considered conservativesubstitutions for one another include: Aromatic: Phenylalanine (F),Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M),Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic:Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q). Seealso Creighton (1984) Proteins, W.H. Freeman and Company, for additionalgroupings of amino acids. Listing of a polypeptide sequence herein, inconjunction with the above substitution groups, provides an expresslisting of all conservatively substituted polypeptide sequences.

In one aspect, the invention provides isolated or recombinantpolypeptides each comprising a sequence having at least 90% sequenceidentity (e.g., at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% amino acidsequence identity) to any one of the polypeptides of FIG. 5. In someinstances the polypeptide exhibits oligoadenylate synthetase activity.

The degree to which a sequence (polypeptide or nucleic acid) is similarto another provides an indication of similar structural and functionalproperties for the two sequences. Accordingly, in the context of thepresent invention, sequences which have a similar sequence to any givenexemplar sequence are a feature of the present invention. In particular,sequences that have percent sequence identities as defined below are afeature of the invention. A variety of methods of determining sequencerelationships can be used, including manual alignment and computerassisted sequence alignment and analysis. A variety of computer programsfor performing sequence alignments are available, or an alignment can beprepared manually by one of skill.

As noted above, the sequences of the polypeptides and nucleic acidsemployed in the subject invention need not be identical, but can besubstantially identical to the corresponding sequence of a polypeptideof the invention or nucleic acid of the invention. For example,polypeptides of the invention can be subject to various changes, such asone or more amino acid insertions, deletions, and/or substitutions,either conservative or non-conservative, including where, e.g., suchchanges might provide for certain advantages in their use, such as, intheir therapeutic or prophylactic use or administration or diagnosticapplication. The nucleic acids of the invention can also be subject tovarious changes, such as one or more substitutions of one or morenucleic acids in one or more codons such that a particular codon encodesthe same or a different amino acid, resulting in either a silentvariation (as defined herein) or non-silent variation, or one or moredeletions of one or more nucleic acids (or codons) in the sequence. Thenucleic acids can also be modified to include one or more codons thatprovide for optimum expression in an expression system (e.g., bacterialor mammalian), while, if desired, said one or more codons still encodethe same amino acid(s). Such nucleic acid changes might provide forcertain advantages in their therapeutic or prophylactic use oradministration, or diagnostic application. The nucleic acids andpolypeptides can be modified in a number of ways so long as theycomprise a sequence substantially identical (as defined below) to asequence in a respective nucleic acid or polypeptide of the invention.

The term “identical” or “identity,” in the context of two or morenucleic acid or polypeptide sequences, refers to two or more sequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same, when compared and aligned for maximumsimilarity, as determined using a sequence comparison algorithm or byvisual inspection.

The “percent sequence identity” (“% identity”) of a subject sequence toa reference (i.e. query) sequence means that the subject sequence isidentical (i.e., on an amino acid-by-amino acid basis for a polypeptidesequence, or a nucleotide-by-nucleotide basis for a polynucleotidesequence) by a specified percentage to the query sequence over acomparison length.

Site Directed Mutagenesis to Create the Polypeptides of the Invention

The polypeptides of the present invention can be engineered using anystandard method of site-directed mutagenesis. The nucleic acid sequencescorresponding to the polypeptides of the invention are synthetized usingspecific oligonucleotide primers and a high fidelity DNA polymerase. Thetarget sequence is contained on a double stranded plasmid isolated froma methylation-competent E. coli strain. Complimentary oligonucleotidescontaining the desired mutation are synthesized and purified usingpolyacrylamide gel electrophoresis. A thermal cycler is used to controlthe temperature for alternating cycles of denaturation of the doublestranded plasmid template (94° C. for 30 seconds), annealing of theoligonucleotide primers (55° C. for 1 minute), and extension of theprimers with a high fidelity polymerase (68° C. for 1 minute/kb ofplasmid length). After approximately 15 cycles, the mixture of newlysynthetized and input DNA are treated with a restriction enzyme specificfor methylated residues (Dpn I) to digest the parental plasmid. Theresulting DNA is introduced into chemically or electrically competentbacterial strains for screening and isolation of plasmids containing thedesired mutation. Plasmid DNA is isolated from the transformants andscreened via fluorescent dye-terminator sequencing to confirm the mutantsequence.

Bulk Drug Product Expression, Fermentation, and Purification

An E. coli strain containing a lysogen of λDE3, and therefore carrying achromosomal copy of the T7 RNA polymerase gene under the control of thelacUV5 promoter, is transformed with a bacterial expression vectorcontaining an IPTG-inducible promoter encoding a nucleic acid sequencecorresponding to one or more of the polypeptides of the presentinvention. Cultures are grown in luria broth medium supplemented with 34μg/mL chloroamphenicol and 15 μg/mL kanamycin at 37° C. When the OD600reaches >0.4, the temperature is reduced to 18° C. and the cells areinduced with 0.5 mM IPTG for 17 hours. The bacterial cells are thenresuspended in buffer containing 50 mM NaH₂PO₄, pH 8, 300 mM NaCl, 20 mMimidazole, 10% glycerol, 0.1% NP40, 2 mM DTT and protease inhibitors(VWR), lysed in a Gaulin homogenizer, and centrifuged to remove celldebris before protein purification.

In one embodiment, purification of the polypeptides of the presentinvention can be achieved using a polyhistidine tag at theamino-terminus. A nickel column is used in affinity purifications ofpolyhistidine tags, with, for example, a 5 mL column being utilized forlysate generated by 4 L of E. coli. The lysate is loaded onto the columnand then washed with Buffer A (50 mM NaH₂PO₄, 300 mM NaCl, 30% glycerol,20 mM imidazole, 2 mM DTT at pH 7.5). A step elution to 7% Buffer B (50mM NaH₂PO₄, 300 mM NaCl, 30% glycerol, 2 M imidazole, 2 mM DTT at pH6.8), for 3.2 column volumes is then carried out. A gradient to 100%Buffer B over 3 column volumes is then carried out. The polypeptide ofthe present invention can then be gel-filtered into Buffer C (50 mMNaH₂PO₄, 150 mM NaCl, 40% glycerol, 1 mM EDTA, 2 mM DTT at pH 6.8) andloaded onto a cation exchange column for further purification. After theprotein is loaded, the column is washed with Buffer C followed by a stepelution to 75% of Buffer D (50 mM NaH₂PO₄, 1 M NaCl, 40% glycerol, 1 mMEDTA, 2 mM DTT at pH 6.8), then a 5 column volume gradient to 100%Buffer D. The protein is then gel filtered into Buffer E (50 mM NaH₂PO4,300 mM NaCl, 40% glycerol, 1 mM EDTA, 2 mM DTT at pH 6.8) and stored at−20° C.

Different embodiments of the polypeptides of the invention, includingbut not limited to: those lacking a polyhistidine tag, those possessinga polyarginine tag, those with reduced cysteine content, those withamino acid sequence variations designed to make the drug candidate morethermally stable, those with modifications to enhance or reduce aparticular activity of the drug candidate, may require alternativepurification strategies. Embodiments of the polypeptide drug candidatelacking a polyhistidine tag, for example, may be directly applied to acation exchange column. Additional steps, for example the use ofhydrophobic interaction chromatography, may be utilized by taking theprotein in Buffer F (50 mM NaH₂PO₄, 300 mM NaCl, 1 M (NH₄)₂SO₄, 30%glycerol, 1 mM EDTA, 2 mM DTT at pH 6.8) and running a 10 column volumegradient to 100% Buffer E. Other affinity columns or sizing columns maybe used to purify different embodiments of the polypeptide drugcandidates.

Alternative techniques may also be used for exchange of buffers,concentration of the drug candidates and purification of the drugcandidates. These could include, but are not limited to,ultrafiltration, tangential flow filtration and diafiltration for theconcentration of the drug candidate and for exchange of buffers.Techniques such a precipitation of the drug candidates by (NH₄)₂SO₄ orsome other chemical agent may also be used. Denaturing the drugcandidate in urea or some other denaturant and refolding it may also beused.

The polypeptides of the present invention are stabilized by excipientscontaining salts; solutions stable at 300 mM NaCl can begin toprecipitate at 150 mM NaCl. For this reason excipient mixtures willfavor these stabilizing salt concentrations, which could include but arenot limited to sodium phosphate, sodium chloride, calcium chloride, andmagnesium chloride.

The addition of amino acid-based excipients such as arginine have provento be stabilizing to the polypeptides of the present invention. A 10%solution of sucrose allows the polypeptides of the invention to bestable at 1 mg/mL, the addition of 2% w/v arginine allows someembodiments of the polypeptides to be stable at 3 mg/mL. For thisreason, other amino acid based compounds, including but not limited tohistidine, glutamine, glycine and human albumin, may be used asexcipients.

The addition of excipients such as glycerol is stabilizing topolypeptides of the present invention. For example, in one embodiment, apolypeptide has a maximum concentration with 10% glycerol (v/v) of 1mg/mL; while at 40% glycerol, the drug candidates are stable up to 12mg/mL. Excipient mixtures containing compounds with similar chemicalproperties are envisioned that include but are not limited to polyolssuch as mannitol, xylitol and sorbitol. Disaccharides such as sucrosehave been found to be stabilizing at 10% w/v; other disaccharidesincluding but not limited to maltose and trehalose can also be used.Monosaccharides can also be used in the present invention. Polysorbates,polyethyleneglycols and similar compounds can also be used to practicethe present invention.

As one skilled in the art will recognize, the use of antioxidants andpreservatives may also be used to ensure stability of the polypeptidesduring storage. Antioxidants, including but not limited to sodiumcitrate, may be stabilizing for long term storage of the polypeptides ofthe invention. Preservatives, including but not limited to, benzylalcohol may also be stabilizing to the polypeptides during storage andmay be used in final excipient mixtures.

Measurement of Oligoadenylate Synthetase Activity of Polypeptides

The oligoadenylate synthetase activities of the polypeptides of theinvention are measured according to previously published methods(Justesen, J., et al. Nuc Acids Res. 8:3073-3085, 1980). Briefly,protein is activated with 200 μg/ml polyinosinic:polycytidylic acid inbuffer containing 20 mM Tris-HCl, pH 7.8, 50 mM Mg(OAc)₂, 1 mM DTT, 0.2mM EDTA, 2.5 mM ATP, α[³²P]ATP, 0.5 mg/ml BSA, and 10% glycerol. Thereaction proceeds at 37° C. for 30 minutes to 24 hours and is terminatedby heating to 90° C. for 3 minutes. 2-4 μl of the reaction mixture isspotted onto a PEI-cellulose thin layer plate. After drying, the plateis developed with 0.4 M Tris-HCl, 30 mM MgCl₂, pH 8.7. The plate isdried and visualized by phosphorimager analysis. Alternatively, thereaction mixture can be further incubated with 0.05 U/μl calf intestinalphosphatase to remove the terminal phosphate. Thin layer chromatographicseparation is achieved using a 0.76 M KH₂PO₄, pH 3.6 developing buffersystem. The plate is then dried and visualized by phosphorimageranalysis.

Measurement of Antiviral Activity of Polypeptides

The ability of the polypeptides of the present invention to protectcultured cells from cytotoxic viruses is demonstrated using a murineencephalomyocarditis virus (EMCV, ATCC strain VR-129B) infection model.Other in vitro virus infection models include but are not limited toflaviviruses such as bovine diarrheal virus, West Nile Virus, and GBV-Cvirus, other RNA viruses such as respiratory syncytial virus, and theHCV replicon systems (e.g. Blight, K. J., et al. 2002. J. Virology,76:13001-13014). Any appropriate cultured cell competent for viralreplication can be utilized in the antiviral assays.

Human Huh7 hepatoma cells are seeded at a density of 1×10⁴ cells/well in96 well culture plates and incubated overnight in complete medium (DMEMcontaining 10% fetal bovine serum). The following morning, the media isreplaced with complete medium containing 0-10 μM protein or equivalentamounts of protein dilution buffer. When desired, alpha-interferon isadded at a concentration of 100 IU/ml. Cells are pretreated for 2-8hours preceding viral infection. After pretreatment, an equal volume ofmedium containing dilutions of EMC virus in complete medium is added tothe wells. In the experiments described herein, a range of 50-500 plaqueforming units (pfu) is added per well.

Viral infection is allowed to proceed overnight (approximately 18hours), and the proportion of viable cells is calculated using anyavailable cell viability or cytotoxicity reagents. The results describedherein are obtained using a cell viability assay that measuresconversion of a tetrazolium compound[3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] to a colored formazan compound in viable cells. Theconversion of MTS to formazan is detected in a 96-well plate reader atan absorbance of 492 nm. The resulting optical densities either areplotted directly to estimate cell viability or are normalized bycontrol-treated samples to calculate a percentage of viable cells aftertreatment.

Polypeptide Pegylation; Sulthydryl

Conjugation of polyethylene glycol (PEG) to the polypeptides of theinvention was achieved by mixing diothiothreitol (DTT)-free purifiedpolypeptide with activated mPEG-MAL (Nektar Therapeutics) at a 0.5-10:1molar ratio. The reaction proceeded at room temperature for 5 min-2hours and was quenched by the addition of 2 mM DTT. Conjugation occurredat multiple cysteine sites using linear 20 kDa and branched 40kDa PEGs.Non-pegylated forms and forms containing one or more PEG can beseparated from each other using a variety of chromatographicmethodologies as known to those skilled in the art. In exemplaryembodiments of the present invention, ion exchange columns, hydrophobicinteractions columns, gel filtration and size exclusion chromatography,each alone or in combination with one another, can be utilized forisolation of the different PEG forms.

Polypeptide Pegylation; N-Terminal

Polypeptides of the invention can be peglyated at the N-terminal amine.To polypeptides in 50 mM NaH₂PO₄, 300 mM NaCl, 30% glycerol, 1 mM EDTA,2 mM DTT at pH 5 containing 20 mM sodium cyanobororohydride and stirringin an ice bath are added a 5-fold excess of mPEG butyrALD-40K. Thereaction is allowed to proceed for up to ten hours and then quenched bythe addition of a 50-fold excess of glycine. Reaction products areanalyzed by SDS-PAGE.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Amino Acid Modifications in Non-Human Primate OAS1Proteins

OAS1 genes from non-human primates were sequenced and compared withmutations found in the human OAS1 gene. Such mutations provideadditional insight into evolution of the OAS1 gene and protein.Evolutionarily conserved amino acids suggest sites important, orcritical, for OAS1 function or enzymatic activity. Conversely, OAS1amino acid sites that have recently mutated, for example in humans only,or show a plurality of amino acid substitutions across primates,indicate sites less critical to function or enzymatic activity. Theabundance of mutated sites within a particular motif of the OAS1 proteinare correlated with the tolerance of that functional domain tomodification. Such sites and motifs are optimized to improve proteinfunction or specific activity. Similarly, mutations in genes andproteins with immune or viral defense functions like OAS1 arehypothesized to result from historical challenge by viral infection.Mutations in primate OAS1 proteins are hypothesized to improveanti-viral efficacy on this basis and are opportunities for optimizationof a human therapeutic OAS1 protein.

In an exemplary embodiment, the ancestral primate amino acid for aspecific site within OAS1 may be restored to a human therapeutic form ofthe OAS1 protein to optimize protein specific activity or anti-viralefficacy. In other embodiments, alternative amino acids identified innon-human primate OAS1s, but not necessarily ancestrally conserved, aresubstituted into a human therapeutic form of OAS1 in order to improveprotein specific activity or anti-viral efficacy. DNA and mRNA sequencesthat code for both the native primate proteins as well as primate-humanhybrid forms are novel and have utility. Several examples of theirutility are: as agents to detect their respective DNA or mRNAcounterparts; in expression vectors used in the manufacture oftherapeutic proteins; and in the detection of novel compounds that bindthe respective mRNA. FIG. 1 provides a therapeutic form of OAS1 (SEQ IDNO:1). Modifications to this form in order to provide additionaltherapeutic forms is performed using at least one amino acidmodification as provided in FIG. 2. As described in FIG. 2, a usefulmodification is the removal of the initial methionine from SEQ ID NO:1and other forms of OAS1. Additional modifications are made as indicatedin FIG. 3. The foregoing modifications described in FIGS. 2 and 3 arealso applied to other therapeutic OAS1 isoforms provided in FIG. 5. FIG.3 also provides specific modifications of OAS1 proteins that arecarboxyl-terminus homologous to Genbank accession NP_(—)002525.1 (forexample, SEQ ID NO:3), specific modifications of OAS1 proteins that arecarboxyl-terminus homologous to Genbank accession NP_(—)0058132.1 (forexample, SEQ ID NO:2) and specific modifications of OAS1 proteins thatare carboxyl-terminus homologous to Genbank accession NP_(—)001027581.1(for example, SEQ ID NO:4). Listed in FIG. 4 are exemplary mutationsidentified in non-human primates including gorilla, chimpanzee,orangutan, and macaque. FIG. 5 lists additional human and non-humanprimate OAS1 isoforms that are useful for the diagnostic and therapeuticpurposes of the present invention as well as particular primatemutations described by the present invention.

Example 2 Preparation and Sequencing of cDNA

Total cellular RNA is purified from cultured lymphoblasts or fibroblastsfrom the patients having the hepatitis C resistance phenotype. Thepurification procedure is performed as described by Chomczynski, et al.,Anal. Biochem., 162:156-159 (1987). The cells are homogenized in 10milliliters (ml) of a denaturing solution containing 4.0M guanidinethiocyanate, 0.1M Tris-HCl at pH 7.5, and 0.1M beta-mercaptoethanol toform a cell lysate. Sodium lauryl sarcosinate is then admixed to a finalconcentration of 0.5% to the cell lysate after which the admixture wascentrifuged at 5000× g for 10 minutes at room temperature. The resultantsupernatant containing the total RNA is layered onto a cushion of 5.7Mcesium chloride and 0.01M EDTA at pH 7.5 and is pelleted bycentrifugation. The resultant RNA pellet is dissolved in a solution of10 mM Tris-HCl at pH 7.6 and 1 mM EDTA (TE) containing 0.1% sodiumdocecyl sulfate (SDS). After phenolchloroform extraction and ethanolprecipitation, the purified total cellular RNA concentration isestimated by measuring the optical density at 260 nm.

Total RNA prepared above is used as a template for cDNA synthesis usingreverse transcriptase for first strand synthesis and PCR witholigonucleotide primers designed so as to amplify the cDNA in twooverlapping fragments designated the 5′ and the 3′ fragment. Theoligonucleotides used in practicing this invention are synthesized on anApplied Biosystems 381A DNA Synthesizer following the manufacturer'sinstructions. PCR is conducted using methods known in the art. PCRamplification methods are described in detail in U.S. Pat. Nos.4,683,192, 4,683,202, 4,800,159, and 4,965,188, and at least in severaltexts including PCR Technology: Principles and Applications for DNAAmplification, H. Erlich, ed., Stockton Press, New York (1989); and PCRProtocols: A Guide to Methods and Applications, Innis, et al., eds.,Academic Press, San Diego, Calif. (1990) and primers based on thenucleotide sequences encoding the amino acid modified regions asdisclosed herein.

The sequences determined directly from the PCR-amplified DNAs from thepatients with and without HCV infection, are analyzed. The presence of amutation upstream from the coding region of the OAS gene can be detectedin patients who are seronegative for HCV despite repeated exposures tothe virus.

The foregoing specification, including the specific embodiments andexamples, is intended to be illustrative of the present invention and isnot to be taken as limiting. Numerous other variations and modificationscan be effected without departing from the true spirit and scope of theinvention. All patents, patent publications, and non-patent publicationscited are incorporated by reference herein.

What is claimed is:
 1. A method for treating a viral infection caused byinfluenza virus in a mammalian subject in need thereof comprisingproviding to the mammalian subject a therapeutically effective amount ofa therapeutic composition comprising a polypeptide consisting of theamino acid sequence of SEQ ID NO:1 except for an amino acid substitutionor deletion at position 1, thereby treating the viral infection in themammalian subject.
 2. A method for treating a viral infection caused byinfluenza virus in a mammalian subject in need thereof comprisingproviding to the mammalian subject a therapeutically effective amount ofa therapeutic composition comprising a polypeptide consisting of theamino acid sequence of SEQ ID NO:1 except the methionine at position 1is deleted and the amino acid at position 162 is substituted, therebytreating the viral infection in the mammalian subject.
 3. A method fortreating a viral infection caused by influenza virus in a mammaliansubject in need thereof comprising providing to the mammalian subject atherapeutically effective amount of a therapeutic composition comprisinga polypeptide consisting of the amino acid sequence of SEQ ID NO:1except the methionine at position 1 is deleted, the amino acid atposition 162 is glycine and the amino acid at position 104 issubstituted, thereby treating the viral infection in the mammaliansubject.
 4. A method for treating a viral infection caused by West Nilevirus in a mammalian subject in need thereof comprising providing to themammalian subject a therapeutically effective amount of a therapeuticcomposition comprising a polypeptide consisting of the amino acidsequence of SEQ ID NO: 1 except for an amino acid substitution ordeletion at position 1, thereby treating the viral infection in themammalian subject.
 5. A method for treating a viral infection caused byDengue virus in a mammalian subject in need thereof comprising providingto the mammalian subject a therapeutically effective amount of atherapeutic composition comprising a polypeptide consisting of the aminoacid sequence of SEQ ID NO: 1 except for an amino acid substitution ordeletion at position 1, thereby treating the viral infection in themammalian subject.
 6. A method for treating a viral infection caused byHepatitis C virus in a mammalian subject in need thereof comprisingproviding to the mammalian subject a therapeutically effective amount ofa therapeutic composition comprising a polypeptide consisting of theamino acid sequence of SEQ ID NO: 1 except for an amino acidsubstitution or deletion at position 1, thereby treating the viralinfection in the mammalian subject.
 7. A method for treating a viralinfection caused by Respiratory Syncytial Virus (RSV) in a mammaliansubject in need thereof comprising providing to the mammalian subject atherapeutically effective amount of a therapeutic composition comprisinga polypeptide consisting of the amino acid sequence of SEQ ID NO: 1except for an amino acid substitution or deletion at position 1, therebytreating the viral infection in the mammalian subject.